Risk and mechanisms of artificial sweetener-induced metabolic dysfunction-associated steatotic liver disease: Insights from multi-level bioinformatics and in vitro assays

The association between artificial sweeteners (ASs), which are widely used sugar substitutes, and metabolic dysfunction-associated steatotic liver disease (MASLD) remains unclear. In this study, we employed an integrated approach to investigate the potential risk and mechanisms of AS-induced MASLD. Using network toxicology, we predicted 534 potential overlapping targets of common ASs (acesulfame, aspartame, cyclamate, neotame, saccharin, and sucralose) and MASLD. Enrichment analysis revealed that these targets participated in multiple pathways, including metabolic regulation, inflammatory response, and immune modulation. Next, machine learning methods were employed in conjunction with GEO datasets (GSE63067 and GSE89632) to identify the potential core targets. NR4A1 and THBS1 were identified as core targets. Molecular docking confirmed the strong binding affinity between all ASs (except acesulfame) and core targets. Based on the molecular docking results, the dominant target pairs (aspartame-NR4A1, cyclamate-NR4A1, neotame-THBS1, saccharin-THBS1, and sucralose-THBS1) for each AS were selected for validation. The CCK-8 assay was used to determine the maximum AS concentration that maintained > 80% viability in HepG2 cells for in vitro validation. In vitro experiments confirmed the inhibition of NR4A1 and THBS1 gene expression by ASs, as well as their potential to induce intracellular lipid accumulation. In conclusion, this study suggests that ASs may promote the onset and progression of MASLD through multiple pathways. NR4A1 and THBS1 are potential toxicological targets. Our findings offer novel insights into AS toxicity and provide a methodological framework for assessing the risks of other substances.